scholarly journals Divalent ion trapping inside potassium channels of human T lymphocytes.

1989 ◽  
Vol 93 (4) ◽  
pp. 609-630 ◽  
Author(s):  
S Grissmer ◽  
M D Cahalan
Keyword(s):  
Divalent Cations ◽  
Ion Trapping ◽  
K Channel ◽  
K Channels ◽  
Current Decay ◽  
Channel Inactivation ◽  
Cell Recording ◽  
Human T Lymphocyte ◽  
K Current ◽  
A Site

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation.

scholarly journals Potassium channel block by internal calcium and strontium.

1991 ◽  
Vol 97 (3) ◽  
pp. 627-638 ◽  
Author(s):  
C M Armstrong ◽  
Y Palti
Keyword(s):  
Time Course ◽  
External Solution ◽  
High Energy ◽  
K Channel ◽  
Giant Fiber ◽  
Distance Measurements ◽  
K Channels ◽  
High Energy Barrier ◽  
Intracellular Ca ◽  
A Site

We show that intracellular Ca blocks current flow through open K channels in squid giant fiber lobe neurons. The block has similarities to internal Sr block of K channels in squid axons, which we have reexamined. Both ions must cross a high energy barrier to enter the blocking site from the inside, and block occurs only with millimolar concentrations and with strong depolarization. With Sr (axon) or Ca (neuron) inside, IK is normal in time course for voltages less than about +50 mV; but for large steps, above +90 mV, there is a rapid time-dependent block or "inactivation." From roughly +70 to +90 mV (depending on concentration) the current has a complex time course that may be related to K accumulation near the membrane's outer surface. Block can be deepened by either increasing the concentration or the voltage. Electrical distance measurements suggest that the blocking ion moves to a site deep in the channel, possibly near the outer end. Block by internal Ca can be prevented by putting 10 mM Rb in the external solution. Recovery from block after a strong depolarization occurs quickly at +30 mV, with a time course that is about the same as that of normal K channel activation at this voltage. 20 mM Mg in neurons had no discernible blocking effect. The experiments raise questions regarding the relation of block to normal channel gating. It is speculated that when the channel is normally closed, the "blocking" site is occupied by a Ca ion that comes from the external medium.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Different properties of the atrial G protein-gated K+ channels activated by extracellular ATP and adenosine

1995 ◽  
Vol 269 (4) ◽  
pp. H1349-H1358 ◽  
Author(s):  
C. Fu ◽  
A. Pleumsamran ◽  
U. Oh ◽  
D. Kim
Keyword(s):  
G Protein ◽  
Single Channel ◽  
Extracellular Atp ◽  
K Channel ◽  
Affinity Constant ◽  
Channel Activity ◽  
K Channels ◽  
Atrial Cells ◽  
K Current ◽  
Inside Out

Extracellular ATP (ATPo) and adenosine activate G protein-gated inwardly rectifying K+ currents in atrial cells. Earlier studies have suggested that the two agonists may use separate pathways to activate the K+ current. Therefore, we examined whether the K+ channels activated by the two agonists have different properties under identical ionic conditions. In cell-attached patches, K+ channels activated by 100 microM ATP in the pipette had a single-channel conductance and mean open time of 32.0 +/- 0.2 pS and 0.5 +/- 0.1 ms, respectively, compared with 31.3 +/- 0.3 pS and 0.9 +/- 0.1 ms for the K+ channels activated by adenosine (140 mM KCl). With ATPo as the agonist, the K+ channel activity in cell-attached patches was approximately threefold lower than that in inside-out patches with 100 microM GTP in the bath. Applying ATP to the cytoplasmic side of the membrane (ATPi) produced a biphasic concentration-dependent effect on channel activity: an increase at low [mean affinity constant (K0.5) = 190 microM] and a decrease at high (K0.5 = 1.3 mM) concentrations. In contrast, with adenosine as the agonist, K+ channel activity in cell-attached patches was approximately fourfold greater than that in inside-out patches with 100 microM GTP in the bath. In inside-out patches, ATPi only augmented the K+ channel activity (K0.5 = 32 microM). These results show that although both ATPo and adenosine activate kinetically similar K+ channels in atrial cells, the channels are regulated differently by intracellular nucleotides.

Intracellular H+ inhibits a cloned rat kidney outer medulla K+ channel expressed in Xenopus oocytes

1995 ◽  
Vol 268 (5) ◽  
pp. C1173-C1178 ◽  
Author(s):  
T. D. Tsai ◽  
M. E. Shuck ◽  
D. P. Thompson ◽  
M. J. Bienkowski ◽  
K. S. Lee
Keyword(s):  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Ph Sensitivity ◽  
Resting Potential ◽  
K Channel ◽  
K Current ◽  
A Site ◽  
The Relationship ◽  
Inwardly Rectifying ◽  
External Ph

The pH sensitivity of a cloned rat kidney K+ channel, ROMK1, was examined after expression in Xenopus oocytes. Membrane currents and intracellular pH (pHi) were concomitantly monitored by the two-microelectrode voltage-clamp technique and a pH-sensitive microelectrode. Oocytes injected with ROMK1 cRNA developed a hyperpolarized resting potential of -98.7 +/- 0.98 mV and a slightly inwardly rectifying Ba(2+)-sensitive K+ current. Lowering external pH from 7.4 to 6.7 using membrane-permeable acetate buffer reduced measured pHi from 7.2 to 6.6 and reduced the ROMK1 current by 80%. The H+ blockade of ROMK1 currents was voltage independent. The relationship between ROMK1 slope conductance and pHi fitted to a titration curve suggested binding of four H+ to a site with a pK of 6.79. Extracellular acidification from pH 7.4 to 6.0 using membrane-impermeable biphthalate buffer had no effect on the ROMK1 current. The pH sensitivity of the ROMK1 channel is similar to that reported for a small-conductance native kidney K+ channel.

scholarly journals Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation

2004 ◽  
Vol 377 (3) ◽  
pp. 569-578 ◽  
Author(s):  
Fabien VAN COPPENOLLE ◽  
Roman SKRYMA ◽  
Halima OUADID-AHIDOUCH ◽  
Christian SLOMIANNY ◽  
Morad ROUDBARAKI ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Proliferation ◽  
Tyrosine Kinase ◽  
Current Amplitude ◽  
K Channel ◽  
Lncap Cells ◽  
Activity Increase ◽  
K Channels ◽  
Long Form ◽  
K Current

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K+ channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K+ current amplitude and the single K+-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K+ channel stimulation. PRL enhances p59fyn phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59fyn antibody is present in the patch pipette, PRL no longer increases K+ current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K+ channel inhibitors α-dendrotoxin and tetraethylammonium. Thus, as K+ channels are known to be involved in LNCaP cell proliferation, we suggest that K+ channel modulation by PRL, via p59fyn pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.

Acetylcholine potentiates acetylcholine-induced increases in K+ current in cat atrial myocytes

1995 ◽  
Vol 268 (3) ◽  
pp. H1313-H1321 ◽  
Author(s):  
Y. G. Wang ◽  
S. L. Lipsius
Keyword(s):  
Reversal Potential ◽  
Membrane Conductance ◽  
K Channel ◽  
Channel Blockers ◽  
Atrial Myocytes ◽  
Initial Exposure ◽  
Recovery Interval ◽  
Cell Recording ◽  
K Current ◽  
K Currents

A nystatin-perforated patch whole cell recording method was used to study the effects of acetylcholine (ACh) on ACh-induced K+ currents in atrial myocytes isolated from cat hearts. The general protocol involved an initial 4-min exposure to ACh (ACh1), followed by a 4-min washout in ACh-free Tyrode solution and then a second 4-min ACh exposure (ACh2). Voltage ramps (40 mV/s) between -130 and +30 mV were used to assess changes in total membrane conductance. ACh2 (10 microM) induced an increase in K+ conductance that was significantly larger than that induced by ACh1 (10 microM) at voltages both negative and positive to the reversal potential. The potentiated current induced by ACh2 reversed at about -80 mV and inwardly rectified at voltages positive to the reversal potential. External Ba2+ (5 mM) or tetraethylammonium (10 mM) abolished all ACh2-induced increases in membrane conductance. The sensitivity to K+ channel blockers, reversal potential, and the rectifying properties indicate that the current potentiated by ACh2 is a K+ current. Atropine (1 microM) blocked all effects of ACh on K+ currents. Potentiation of K+ current by ACh2 required 1) ACh1 concentrations > or = 1 microM, 2) ACh1 duration > or = 2 min, and 3) recovery interval > or = 2 min. We conclude that an initial exposure to ACh potentiates subsequent ACh-induced increases in K+ current. ACh-induced potentiation depends on the concentration and duration of the initial ACh exposure and the recovery interval between consecutive ACh exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ and pH regulation of K+ channels in membrane vesicles of rabbit proximal tubule

1990 ◽  
Vol 258 (6) ◽  
pp. F1634-F1639
Author(s):  
C. Jacobsen ◽  
S. Mollerup ◽  
M. I. Sheikh
Keyword(s):  
Proximal Tubule ◽  
Ph Regulation ◽  
Membrane Vesicles ◽  
Inhibitory Effect ◽  
K Channel ◽  
Maximal Inhibition ◽  
K Channels ◽  
A Site ◽  
Pars Convoluta

The effect of Ca2+ and pH on the renal epithelial K+ channel was investigated by measuring the Ba2(+)-sensitive 86Rb+ fluxes in membrane vesicles from pars convoluta of rabbit proximal tubule. It was found that the presence of nanomolar concentrations of Ca2+ in the internal compartment (cytoplasmic) of the vesicles ([Ca2+]i) substantially lowered the channel-mediated flux. Ba2(+)-sensitive 86Rb+ uptake was completely blocked by 10 microM [Ca2+]i. This inhibitory effect of Ca2+ was strongly dependent on pH. Thus 0.1 microM [Ca2+]i produced a maximal inhibition of 86Rb+ uptake at pH greater than 7.4 but had no effect at pH less than 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over this range. The data are compatible with the model that Ca2+ blocks K+ channels by binding to a site composed of one or several deprotonated groups. The protonation of any one of these groups prevents Ca2+ from binding to this site but does not by itself block transport.

scholarly journals Dynamics of aminopyridine block of potassium channels in squid axon membrane.

1976 ◽  
Vol 68 (5) ◽  
pp. 519-535 ◽  
Author(s):  
J Z Yeh ◽  
G S Oxford ◽  
C H Wu ◽  
T Narahashi
Keyword(s):  
Time Course ◽  
Membrane Surface ◽  
K Channel ◽  
Dependent Manner ◽  
Squid Axon ◽  
Axon Membrane ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current ◽  
Kinetics Of

Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequency-dependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley n-type gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.

Local Anesthetics Modulate Neuronal Calcium Signaling through Multiple Sites of Action

2003 ◽  
Vol 98 (5) ◽  
pp. 1139-1146 ◽  
Author(s):  
Fang Xu ◽  
Zayra Garavito-Aguilar ◽  
Esperanza Recio-Pinto ◽  
Jin Zhang ◽  
Thomas J. J. Blanck
Keyword(s):  
Local Anesthetics ◽  
K Channel ◽  
Na Channels ◽  
Channel Blockade ◽  
K Channels ◽  
Voltage Dependent ◽  
Sites Of Action ◽  
A Site ◽  
Ca2 Channels

Background Local anesthetics (LAs) are known to inhibit voltage-dependent Na+ channels, as well as K+ and Ca2+ channels, but with lower potency. Since cellular excitability and responsiveness are largely determined by intracellular Ca2+ availability, sites along the Ca2+ signaling pathways may be targets of LAs. This study was aimed to investigate the LA effects on depolarization and receptor-mediated intracellular Ca2+ changes and to examine the role of Na+ and K+ channels in such functional responses. Methods Effects of bupivacaine, ropivacaine, mepivacaine, and lidocaine (0.1-2.3 mm) on evoked [Ca2+](i) transients were investigated in neuronal SH-SY5Y cell suspensions using Fura-2 as the intracellular Ca2+ indicator. Potassium chloride (KCl, 100 mm) and carbachol (1 mm) were individually or sequentially applied to evoke increases in intracellular Ca2+. Coapplication of LA and Na+/K+ channel blockers was used to evaluate the role of Na+ and K+ channels in the LA effect on the evoked [Ca2+](i) transients. Results All four LAs concentration-dependently inhibited both KCl- and carbachol-evoked [Ca2+](i) transients with the potency order bupivacaine > ropivacaine > lidocaine >/= mepivacaine. The carbachol-evoked [Ca2+](i) transients were more sensitive to LAs without than with a KCl prestimulation, whereas the LA-effect on the KCl-evoked [Ca2+](i) transients was not uniformly affected by a carbachol prestimulation. Na+ channel blockade did not alter the evoked [Ca2+](i) transients with or without a LA. In the absence of LA, K+ channel blockade increased the KCl-, but decreased the carbachol-evoked [Ca2+](i) transients. A coapplication of LA and K+ channel blocker resulted in larger inhibition of both KCl- and carbachol-evoked [Ca2+](i) transients than by LA alone. Conclusions Different and overlapping sites of action of LAs are involved in inhibiting the KCl- and carbachol-evoked [Ca2+](i) transients, including voltage-dependent Ca2+ channels, a site associated with the caffeine-sensitive Ca2+ store and a possible site associated with the IP(3)-sensitive Ca2+ store, and a site in the muscarinic pathway. K+ channels, but not Na+ channels, seem to modulate the evoked [Ca2+](i) transients, as well as the LA-effects on such responses.

Halothane and Isoflurane Decrease the Open State Probability of Potassium sup + Channels in Dog Cerebral Arterial Muscle Cells 

1995 ◽  
Vol 82 (2) ◽  
pp. 479-490 ◽  
Author(s):  
Hanna Eskinder ◽  
Debebe Gebremedhin ◽  
Joseph G. Lee ◽  
Nancy J. Rusch ◽  
Franjo D. Supan ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Muscle Cells ◽  
Volatile Anesthetics ◽  
State Probability ◽  
K Channel ◽  
Dependent Manner ◽  
External Application ◽  
Pipette Solution ◽  
K Channels ◽  
K Current

Background Both halothane and isoflurane evoke cerebral vasodilation. One of the potential mechanisms for arterial vasodilation is enhanced K+ efflux resulting from an increased opening frequency of membrane K+ channels. The current study was designed to determine the effects of volatile anesthetics on K+ channel current in single vascular smooth muscle cells isolated from dog cerebral arteries. Methods Patch clamp recording techniques were used to investigate the effects of volatile anesthetics on macroscopic and microscopic K+ channel currents. Results In the whole-cell patch-clamp mode, in cells dialyzed with pipette solution containing 2.5 mM EGTA and 1.8 mM CaCl2, depolarizing pulses from -60 to +60 mV elicited an outward K+ current that was blocked 65 +/- 5% by 3 mM tetraethylammonium (TEA). Halothane (0.4 and 0.9 mM) depressed the amplitude of this current by 18 +/- 4% and 34 +/- 6%, respectively. When 10 mM EGTA was used in the pipette solution to strongly buffer intracellular free Ca2+, an outward K+ current insensitive to 3 mM TEA was elicited. This K+ current, which was reduced 51 +/- 4% by 1 mM 4-aminopyridine, was also depressed by 17 +/- 5% and 29 +/- 7% with application of 0.4 and 0.9 mM halothane, respectively. In cell-attached patches using 145 mM KCl in the pipette solution and 5.2 mM KCl in the bath, the unitary conductance of the predominant channel type detected was 99 pS. External application of TEA (0.1 to 3 mM) reduced the unitary current amplitude of the 99 pS K+ channel in a concentration-dependent manner. The open state probability of this 99 pS K+ channel was increased by 1 microM Ca2+ ionophore (A23187). These findings indicate that the 99 pS channel measured in cell-attached patches was a TEA-sensitive, Ca(2+)-activated K+ channel. Halothane and isoflurane reversibly decreased the open state probability (NPo), mean open time, and frequency of opening of this 99 pS K+ channel without affecting single channel amplitude or the slope of the current-voltage relationship. Conclusions Halothane and isoflurane suppress the activity of K+ channels in canine cerebral arterial cells. These results suggest that mechanisms other than K+ channel opening likely mediate volatile anesthetic-induced vasodilation.

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